Rhona J McVey

Inhibition of FGFR2 and FGFR1 increases cisplatin sensitivity in ovarian cancer

Fibroblast Growth Factors (FGFs) have been implicated in malignant transformation, tumour mitogenesis, angiogenesis and chemoresistance. The aim of this study was to determine which FGFs and FGFRs play functional roles in epithelial ovarian cancer. Restriction enzyme analysis of mRNA revealed that transformation was associated with a switch in FGFR2 and FGFR3, from the IIIc to the IIIb isoform. There was widespread expression of FGFs, including FGF7, in all tissues but, FGF3 and FGF19 were expressed by malignant cell lines and cancer tissue but were not present in normal tissue. Using FGFR-specific shRNAi we demonstrated that reductions in FGFR2 inhibited proliferation of ovarian cancer cell lines in vitro (>50%, p < 0.006), and reduced cisplatin IC50 (>60%, p < 0.0001). Cell cycle analysis revealed increased cisplatin sensitivity was associated with increased G2/M arrest and increased apoptosis. FGFR2 shRNAi reduced growth rates of ovarian tumour xenografts by 20% (p<0.006) and when combined with cisplatin caused a 40% reduction in proliferation rates (p<0.007). In contrast, RNAi-induced reductions in FGFR1 increased SKOV3 cell numbers, with associated changes in cell cycle but had no effect on ES2 cells. However, the cisplatin IC50 was reduced (>50%, p < 0.0001) by FGFR1 shRNAi in both cell lines and there was increased apoptosis (46-50%) compared with control cells (35%) (p < 0.004). Together our data suggest that combining FGFR2 inhibitors with platinum-containing cytotoxic agents for the treatment of epithelial ovarian cancer may yield increased anti-tumour activity. However, data on the inhibition of FGFR1 suggest that broad spectrum FGFR inhibitors may have unexpected effects on proliferation.

Heparan sulphate synthetic and editing enzymes in ovarian cancer.

Several angiogenic growth factors including fibroblast growth factors 1 and 2 (FGF1 and FGF2) depend on heparan sulphate (HS) for biological activity. We previously showed that all cellular elements in ovarian tumour tissue synthesised HS but biologically active HS (i.e. HS capable of binding FGF2 and its receptor) was confined to ovarian tumour endothelium. In this study, we have sought to explain this observation. Heparan sulphate sulphotransferases 1 and 2 (HS6ST1 and HS6ST2) attach sulphate groups to C-6 of glucosamine residues in HS that are critical for FGF2 activation. These enzymes were strongly expressed by tumour cells, but only HS6ST1 was found in endothelial cells. Immunostaining with the 3G10 antibody of tissue sections pretreated with heparinases indicated that HS proteoglycans were produced by tumour and endothelial cells. These results indicated that, in contrast to the endothelium, HS produced by tumour cells may be modified by cell-surface heparanase (HPA1) or endosulphatase (SULF). Protein and RNA analysis revealed that HPA1 was strongly expressed by ovarian tumour cells in eight of ten specimens examined. HSULF-1, which removes specific 6-O-sulphate groups from HS, was abundant in tumour cells but weakly expressed in the endothelium. If this enzyme was responsible for the lack of biologically active HS on the tumour cell surface, we would expect exogenous FGF2 binding to be preserved; we showed previously that this was indeed the case although FGF2 binding was reduced compared to the endothelium and stroma. Thus, the combined effects of heparanase and HSULF could account for the lack of biologically active HS in tumour cells rather than deficiencies in the biosynthetic enzymes.

Regulation of fibroblast growth factor-2 activity by human ovarian cancer tumor endothelium

Fibroblast growth factor-2 (FGF-2) is a potent angiogenic cytokine that is dependent on heparan sulfate for its biological activity. We have investigated the relationship among heparan sulfate, FGF-2, and the signal-transducing receptors in human, advanced-stage, serous ovarian adenocarcinoma. Using a unique molecular probe, FR1c-Ap, which consisted of a soluble FGF receptor 1 isoform lllc covalently linked to an alkaline phosphatase moiety, the distribution of heparan sulfate that had the ability to support the formation of a heparan sulfate/FGF-2/FGFR1 isoform IIIc alkaline phosphatase heparan sulfate construct complex was determined. This may be taken as a surrogate marker for the distribution of biologically active heparan sulfate and was distributed predominantly in endothelial cells and stroma but was absent from adenocarcinoma cells. In situ hybridization revealed the expression of FGFR1 mRNA in the endothelium and reverse transcription-PCR confirmed the presence of FGFR1 isoform IIIc but not isoform IIIb. The presence of FGF-2 around tumor endothelium was detected through immunohistochemistry. Double-staining techniques showed that heparan sulfate was found predominantly at the basal aspect of the endothelium and suggested that syndecan-3 might function as one of the proteoglycans involved in FGF-2 signaling in the endothelium. The data suggest that the entire extracellular signaling apparatus, consisting of FGF-2, biologically active heparan sulfate, and FGFRs capable of responding to FGF-2, is present in ovarian cancer endothelium, thereby highlighting the cytokine and its cognate receptor as potential targets for the antiangiogenic treatment of this disease.

Measuring the biological effect of presurgical metformin treatment in endometrial cancer

Background:Preclinical studies in endometrial cancer (EC) show that metformin reduces cellular proliferation by PI3K-AKT-mTOR inhibition. We tested the hypothesis that short-term presurgical metformin reduces cellular proliferation in atypical endometrial hyperplasia (AEH) and endometrioid EC, and assessed the feasibility of using phosphorylated PI3K-AKT-mTOR proteins as tissue end points.Methods:Women with AEH or EC received metformin 850 mg twice a day or no drug in the presurgical window between diagnosis and hysterectomy. Before and after the window, tissue samples were obtained; serum markers of insulin resistance (e.g. homeostasis model of assessment of insulin resistance index) were determined; and anthropometrics measured (e.g. BMI). Cell proliferation (Ki-67) and PI3K-AKT-mTOR phosphostatus were assessed by immunohistochemistry and scored blinded to treatment.Results:Twenty-eight metformin-treated and 12 untreated patients, well matched for age and BMI, completed the study. Metformin treatment (median 20 days, range 7–34) was associated with a 17.2% reduction in tumour Ki-67 (95% CI −27.4, −7.0, P=0.002), in a dose-dependent manner. Tumour PI3K-AKT-mTOR protein phosphostatus varied but the effects were not significant after adjusting for changes in controls.Conclusions:Short-term metformin was associated with reduced Ki-67 expression in EC. Changes in tumour PI3K-AKT-mTOR protein phosphostatus were seen in both groups. Future studies should address the variability attributed to different sampling techniques including devascularisation of the uterus at hysterectomy.

Transvaginal biopsy in the diagnosis of ovarian cancer

Laparotomy and debulking surgery followed by chemotherapy have been the treatment of choice in late stage ovarian carcinoma. Developments in the chemotherapeutic management of ovarian cancer have resulted in a change in opinion as to the optimal management of this disease. Many patients are now receiving initial chemotherapy and trials are in place to compare up front and adjuvant surgery. Tissue diagnosis is required prior to commencing chemotherapy. This article describes one method for accurately obtaining a tumour biopsy. A retrospective case note review of 14 women with a provisional diagnosis of ovarian carcinoma who underwent transvaginal biopsy of their pelvic disease is described. Only 7/12 cases with a positive biopsy had a definite diagnosis of ovarian cancer. The procedure was found to be safe and well tolerated.

A presurgical window-of-opportunity study of metformin in obesity-driven endometrial cancer.

BACKGROUND Metformin use is associated with reduced cancer risk in several observational studies of patients with type 2 diabetes. Results from preclinical studies in endometrial cancer show that metformin reduces cellular proliferation by inhibition of the PI3K-AKT-mTOR pathway. We tested the hypothesis that metformin would reduce cellular proliferation in vivo in atypical endometrial hyperplasia and endometrial endometrioid adenocarcinoma. METHODS We recruited women attending gynaecological oncology clinics in Manchester, UK, with atypical endometrial hyperplasia or endometrial endometrioid adenocarcinoma. Women received metformin (850 mg twice daily) or no drug (control) during the 1-4 week presurgical window between cancer diagnosis and hysterectomy according to patient preference. Paired blood and tumour samples were obtained at recruitment and hysterectomy. Cellular proliferation was assessed by Ki-67 proliferation index. Automated scoring on two separate occasions provided consistent replicate scores (SD <10%). This study is registered with the ISRCTN register, number ISRCTN81570194. FINDINGS Samples from 40 women have been analysed (28 metformin-treated [median age 64 years, IQR 58-69]; 12 control [70, 64-70]). 24 of the patients (60%) were obese. 22 patients (55%) had either undiagnosed diabetes (fasting glucose >7·0 mmol/L, n=4) or insulin resistance (homoeostatic model assessment of insulin resistance >2·8, n=18). Metformin was taken for a median of 20 days (IQR 17-24), and mild gastrointestinal side-effects were reported by 22 metformin-treated patients. In the metformin-treated group, Ki-67 was 12·9% lower at hysterectomy than at recruitment (95% CI 3·7-22·1, p=0·008) after adjustment for baseline Ki-67, Ki-67 change in controls, age, and body-mass index. No significant changes in phosphorylation of AKT or markers of insulin resistance after adjustment for treatment arm were seen. INTERPRETATION Undiagnosed insulin resistance or diabetes were common in our study population. Short-term presurgical metformin was associated with a reduction in Ki-67 proliferation index. We are now exploring the hypothesis that metformin reduces Ki-67 expression by inducing phosphorylation of AMP-activated kinase and subsequent mTOR proproliferative inhibition, independent of insulin and insulin-like growth factor receptor activation. FUNDING Wellbeing of Women, Wellcome Trust.

Ki-67 in endometrial cancer: Scoring optimization and prognostic relevance for window studies

Ki-67, a marker of cellular proliferation, is increasingly being used in pre-surgical window studies in endometrial cancer as a primary outcome measure. Unlike in breast cancer, however, there are no guidelines standardizing its measurement and its clinical relevance as a response biomarker is undetermined. It is, therefore, imperative that Ki-67 scoring protocols are optimized and its association with patient survival rigorously evaluated, in order to be able to clinically interpret the results of these studies. Using the International Ki-67 in Breast Cancer Working Group guidelines as a basis, whole slide, hot spot and invasive edge scoring protocols were evaluated using endometrial biopsies and hysterectomy specimens from 179 women. Whole sections and tissue microarrays, manual and semi-automated scoring using Definiens Developer software were additionally compared. Ki-67 scores were related to clinicopathological variables and cancer-specific survival in uni- and multivariate analysis. Against criteria of time efficiency, intra- and inter-observer variability and consistency, semi-automated hot spot scoring was the preferred method. Ki-67 scores positively correlated with grade, stage and depth of myometrial invasion (P-values all <0.03). By univariate analysis, higher Ki-67 scores were associated with a significant reduction in cancer-specific survival (P≤0.05); however, this effect was substantially attenuated in the multivariate model. In conclusion, hot spot scoring of whole sections using Definiens is an optimal method to quantify Ki-67 in endometrial cancer window study specimens. Measured this way, it is a clinically relevant marker, though further work is required to determine whether reductions in Ki-67 in neoadjuvant intervention studies translate into improved patient outcome.

Primary extranodal marginal zone B cell lymphoma of the uterus: a case study and review of the literature

Extranodal non-Hodgkins lymphoma (NHL) within the female genital tract is unusual, accounting for 1.5% of extranodal NHL, mostly in the ovaries. Primary NHLs of the uterus and cervix are rare, comprising only 0.54%–0.64% of all extranodal NHLs, most occurring in the cervix.1 Marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) of the uterus is extremely rare, with only seven cases reported in the literature.2–8 We report a rare case of an extranodal marginal zone B cell lymphoma arising from the MALT tissue of the endometrium. A 77-year-old woman presented with utero-vaginal prolapse, urinary frequency and nocturia; she had no B symptoms. Examination revealed a cystocoele and a cervical prolapse. There was no palpable lymphadenopathy or hepatosplenomegally and she underwent vaginal hysterectomy and pelvic floor repair. The uterus and cervix measured 8.3×2.6×2.2 cm. The endometrium was 0.2 cm thick and the myometrium 1.6 cm thick; both appeared normal. Microscopically, the endometrium and superficial myometrium contained a nodular infiltrate of monotonous, small to medium-sized, lymphoid cells (figure 1A). The cells …

Ancillary testing in liquid based cytology to distinguish cervical glandular neoplasia from tuboendometrial metaplasia in a young woman

A 33‐year old woman had a cervical sample taken at colposcopy clinic. Seven years prior to this, at the age of 26, she had had a cytological diagnosis of cervical glandular neoplasia (cytology descriptor indicated cells suspicious of endocervical neoplasia) and severe dyskaryosis. Confirmation and treatment were by LLETZ and knife cone, and, in keeping with England and Wales National Health Service guidelines, this woman was under follow‐up by the colposcopy clinic. Intervening cytological follow‐up included a number of negative cytological samples interspaced with one equivocal report. A recent repeat cytology which was rather cellular contained several hyperchromatic crowded cell groups (HCCGs). Careful examination revealed benign endometrial clusters, LUS, TEM and endocervical cells in strips showing pseudostratification and loss of polarity. Following an agar block, there was positive staining for p16 and Ki‐67 in the abnormal groups whilst the benign TEM cells stained positive for bcl‐2. Diagn. Cytopathol. 2010;38:828–832.

BRCA and lynch syndrome-associated ovarian cancers behave differently

We are delighted to have the opportunity to respond to the letter by Gilks et al. (Gilks et al., 2017). They argue first that historical pathological entities do not map seamlessly to modern day diagnostic categories, which are not only based on expert review by specialist gynecological pathologists but are also increasingly supported by immunohistochemistry (IHC). Second, that the misclassification of biologically indolent tumors as high grade serous (HGS) ovarian cancers may explain the uncharacteristically excellent survival figures we report (Ryan et al., 2017). And third, that our paper may inadvertently lead to the universal screening of HGS ovarian cancers for Lynch syndrome, when this is not indicated. To address the first point, we undertook contemporary slide review and supporting IHC for a subset of our cases (12/37, 30%). In so doing, we were able to compare the original histological subtype with up-to-date expert gynecological pathology review backed up by IHC. Unfortunately, we were unable to retrieve slides or tissue blocks for all women, particularly those whose surgery was performed elsewhere and more than 15 years ago. Table 1 shows the results of our analysis. Pathology review confirmed accurate diagnoses for 10 of the 12 cases (83%), including six endometrioid tumors, one clear cell, one carcinosarcoma, one mixed endometrioid/clear cell tumor and one poorly differentiated carcinoma which was difficult to reliably subtype* (*Fig. 1D). The two discrepant cases were an endometrioid tumor originally misclassified as a ‘serous cystadenocarcinoma’, and a ‘serous papillary carcinoma’, which had cytology of low-to-intermediate grade on review and wild type p53 staining favouring a final classification of low grade serous carcinoma** (**Fig. 1A). Our data are in keeping with those of other case series of Lynch syndrome-associated ovarian cancer, where endometrioid morphology predominates, but clear cell, serous and mixed histotypes are also described (Grindedal et al., 2010; Coppola et al., 2012; Zhai et al., 2008; Rosenthal et al., 2013). Regarding the second point, we speculated that the mismatch repair (MMR) status of the tumors could help differentiate those that had arisen because of mismatch repair deficiency from those that had arisen via a different pathway. As shown in Fig. 1 and Table 1, all of the tumors showed MMR deficiency in keeping with their inherited mutation, including the women whose tumors showed serous morphology. This supports the hypothesis that all have arisen as a consequence of Lynch syndrome. We and others have shown that Lynch syndrome-associated ovarian cancer presents at an earlier age and stage and demonstrates excellent survival rates compared with sporadic ovarian cancers (Moller et al., 2017; Lynch et al., 2009). This may be due to early detection through surveillance, good biology, enhanced tumor immunogenicity and responsiveness to current treatments (Lynch et al., 2009). By contrast, BRCA-associated ovarian cancers have a poor prognosis irrespective of early detection and treatment (Finch et al., 2014). There is no doubt that germline mutations in MMR and BRCA genes are only similar because they both predispose a woman to developing ovarian cancer; in every other respect, they are quite different. Finally, we do not recommend that all HGS ovarian cancers are routinely screened for Lynch syndrome. The prevalence of Lynch syndrome in women with ovarian cancer is low. However, genetic predisposition syndromes should always be considered as an underlying cause for non-